Our research program over the past three decades has been guided by the fundamental tenet that voltage- gated channel proteins are modular in design and that the coupling between the component modules underlies their exquisite sensitivity to voltage and governs their functional diversity. For the new funding period, the thre main goals focus on: 1. The determination of the crystal structure of the channel in an open conformation attempting to understand the role of the sensor module. We will pursue the structure-function program of the pore module by supplementing the experimental conditions used to crystallize it with novel open-conformation stabilizers aiming to decode the open channel structure. 2. The elucidation of the origin of the voltage dependence in terms of the inherent properties of the sensorless pore, and on the regulation conferred by partner modules including a voltage sensor or a mechanical stress sensor. Numerous mutants generated at key locations on the PM structure favor the conductive state of the pore; accordingly, we will attempt to crystallize the most interesting candidates aiming to uncover an open structure. Having established the channel properties of the purified mechanosensitive Piezo proteins reconstituted in lipid bilayers leads us to apply our molecular dissection strategy to identify the structural determinants of the pore module and of the force sensor module. A vast number of chimeras, truncated constructs, and carefully selected mutants will be generated and functionally characterized along the lines previously reported. The ultimate aim is to confer mechanosensitivity to the robust PM scaffold by appending the force sensor module of Piezo proteins. 3. The structural and functional characterization of a newly discovered voltage-activated anion-selective channel (Xv). An immediate goal entails an in-depth functional characterization in terms of selectivity, permeation, and block of the purified full-length Xv and f its sensorless-PM after reconstitution in lipid bilayers. Attention will be directed to establish te voltage-dependence of activation given that Xv may represent a hyperpolarization-activated channel. An urgent task is to crystallize Xv and its PM using the lipid cubic phase approach we successfully applied for KvLm. The procedures we developed over this +30-year interval to investigate structure-function relationships together with newly acquired capability for structure determination augur a realistic, innovative, and productive continuation of our long-term commitment to a mechanistic understanding of voltage sensing in terms of the modular design of voltage-gated channel proteins.